Compositions having inhibited ability to promote platelet aggregation or fibrin clot formation

ABSTRACT

A composition which comprises a substance which is a substituted aromatic and which inhibits the thrombotic or clotting activity of a material, chemically bonded through an --N=N-- grouping to a second aromatic which is chemically bonded at a sulfonyl grouping through a nitrogen containing chemical bond to a material which is susceptible to interaction with a nitrene.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of co-pending application Ser. No.258,016, filed May 30, 1972 and now issued as U.S. Pat. No. 3,888,833.

BACKGROUND OF THE INVENTION

Technological advances in the last few decades have brought themammalian body in intimate contact with a variety of devicesincorporating foreign materials which were not present in the evolutionof their systems. Although many of these devices function as designed, asubstantial number of them suffer from serious drawbacks. This appearsto be particularly true when a device prepared from a foreign materialis in contact with the blood of a mammal. All presently availablematerials which are in contact with the blood for significant periods oftime induce deposition of blood cells and fibrin. This problem hashampered the practical development of artificial organs, extracorporealshunts, renal dialysis systems, catheters, heart-lung machines and thelike. Solution of the problem is also important for in vitro systemssuch as blood collection and storage containers, for example.

Interaction of blood with the surface of a foreign material can resultin two related but distinct processes. The first is a formation of aplatelet thrombus which may lead to a second process, namely,coagulation, particularly in areas of slow blood flow. However,coagulation can occur in the absence of platelet interaction,particularly in in vitro systems.

The platelet thrombosis process is initiated by the adherence of bloodplatelets to the foreign surface with subsequent platelet-plateletinteraction or aggregation leading to formation of a thrombus composedalmost entirely of platelets. In areas of rapid blood flow, such as thecage of an artificial heart valve or in arterial grafts, the primarydifficulty is platelet thrombosis and thromboembolism. Aggregatedplatelets release a factor which may stimulate a second process, bloodcoagulation, leading to formation of a fibrin blood clot. Clot formationis of major importance in areas of slow blood flow such as around thesewing ring of a valve or in venous circulation. Although plateletthrombi do not always result in clot formation, they can producedeleterious effects themselves. Since there is not a generic descriptioncovering both of these processes, a substance which inhibits plateletaggregation will hereinafter be referred to as "antithrombotic" and asubstance which inhibits fibrin clot formation will hereinafter bereferred to as "anticlotting".

Previously, researchers have attacked the problem of deposition of bloodcells and fibrin on the surfaces of materials primarily by attempting toinhibit clot formation. The most common approach has been to coat thesurface or impregnate the material with an anticoagulant, primarilyheparin. Because there is no known method of directly binding heparin tomaterials, the heparin is bound to an intermediate chemical which isalso bound to the particular material.

The binding of the intermediate to the material was done initiallythrough adsorption. Since this surface gradually wore off the material,an ionic type of intermediate became employed more frequently. Thelifetime of this coating was generally longer but it gradually wore off,as well. Recently, the intermediate compound has been covalently boundto the material. However, this covalent bonding is dependent upon areactive group such as gamma propylamine in or interspersed within thematerial, or on radiation grafting.

BRIEF SUMMARY OF THE INVENTION

We have now found a new means of covalently bonding antithrombotic oranticlotting compounds to a material. This bonding is brought about bythe chemical attachment of the antithrombotic or anticlotting substanceto an aromatic sulfonyl nitrene which is chemically bonded to thematerial, apparently through means of a sulfonamide-type bond. Anymaterial which is susceptible to this type of interaction can have itsability to promote platelet aggregation or fibrin clot formationinhibited.

Therefore, it is in accordance with this invention that we havediscovered a method for binding antithrombotic or anticlottingsubstances to a susceptible material involving the use of an aromaticsulfonyl nitrene.

A further aspect of this invention involves the composition of theantithrombotic or anticlotting substance, the aromatic sulfonyl nitrene,and the material; and the method of inhibiting platelet aggregation orfibrin clot formation with these compositions.

A still further aspect of the invention is the aromatic sulfonyl nitreneantithrombotic or anticlotting substance intermediate.

The formation of intermediate compositions of the aromatic sulfonylnitrene and the material is another aspect of this invention.

DETAILED DESCRIPTION OF THE INVENTION

The materials to which antithrombotic or anticlotting substances can beindirectly bound are materials which are susceptible to reaction with anaromatic sulfonyl nitrene. Generally these materials are polymers,either synthetic or natural, with a plurality of carbon-hydrogen bonds.The preferable materials include polyethylene, polypropylene, natural orsynthetic rubbers, a polyester such as Dacron, nylons, polyurethanes,and the like.

The actual compound which is bound to the surface of the material is thearomatic sulfonyl nitrene. The reaction route appears to be theinsertion of the nitrene into a carbon and hydrogen bond of the polymerupon irradiation of an azide, thus forming a sulfonamide. This isillustrated below with a para nitrobenzenesulfonamide. ##STR1##

The aromatic sulfonyl nitrene which can be bound to the surface of thematerial is selected from the group of aromatic sulfonyl nitrenesconsisting of ##STR2## wherein R and R₁ can be the same or different andare selected from the group consisting of hydrogen, halogen, and normalor isomerized alkyl from one to four carbon atoms, inclusive, and##STR3## where R and R₁ are defined as in FIG. 11 above and R₂ is asubstance which inhibits the thrombotic or clotting activity of thematerial and is that portion of a substituted aromatic which coupleswith a diazonium salt. Halogen is fluorine, chlorine, bromine, andiodine. Alkyl is methyl, ethyl, propyl, butyl, and isomers thereof.

Aromatic sulfonyl nitrenes of FIG. 11 are made from sulfonylchlorides byconversion of the sulfonyl chloride to the corresponding sulfonylazide.The azide is converted to the corresponding nitrene by irradiation.Illustrative sulfonylchloride starting materials for the nitrenes ofFIG. 11 are the following compounds:

2-bromo-5-nitrobenzenesulfonylchloride

2-chloro-4-nitrobenzenesulfonylchloride

2-chloro-5-nitrobenzenesulfonylchloride

4-chloro-2-nitrobenzenesulfonylchloride

4-chloro-3-nitrobenzenesulfonylchloride

4-fluoro-3-nitrobenzenesulfonylchloride

4-propyl-2-nitrobenzenesulfonylchloride

4-tertbutyl-2-nitrobenzenesulfonylchloride

4-propyl-2-chloro-5-nitrobenzenesulfonylchloride

4-nitro-o-toluenesulfonylchloride

5-nitro-o-toluenesulfonylchloride

3-nitro-o-toluenesulfonylchloride

5-chloro-3-nitro-o-toluenesulfonylchloride

2,4-dichloro-5-nitrobenzenesulfonylchloride

4-propyl-6-methyl-2-nitrobenzenesulfonylchloride

Illustrative compounds of FIG. 111 are the aromatic sulfonyl nitrenes ofFIG. 11 with the R₂ compound being that portion of the followingillustrative substituted aromatics which couple with a diazonium salt:

8-amino-1-naphthol-5,7-disulfonic acid

1-amino-2-naphthol-4-sulfonic acid

4,5-dihydroxynaphthalene-2,7-disulfonic acid

2,3-dihydroxynaphthalene-6-sulfonic acid

7-amino-1-naphthol-3,6-disulfonic acid

8-amino-1-naphthol-3,6-disulfonic acid

1-naphthol-3,6-disulfonic acid

2-naphthol-3,6-disulfonic acid

1-naphthol-4-sulfonic acid

2-naphthol-7-sulfonic acid

α-methyl-2-fluoro-2'-hydroxy-4-biphenylacetic acid

α-methyl-2-fluoro-4'-hydroxy-4-biphenylacetic acid

The preferred substituted aromatic is 8-amino-1-naphthol-5,7-disulfonicacid.

The preferred R and R₁ substituents of FIG. 11 and FIG. 111 compoundsinclude compounds where R is hydrogen, chloride, bromide, and alkyl fromone to three carbon atoms, inclusive.

The most preferred are those compounds where R and R₁ are hydrogen andthe nitro group is meta or para.

The compounds and compositions of the invention can be prepared bymethods known in the art. The synthesis of FIG. 11 and FIG. 111compounds, aromatic sulfonylnitrene-material intermediate compositionsand final compositions of the material-aromatic sulfonylnitrene-antithrombotic or anticlotting substance will be illustratedemploying the meta-nitrobenzenesulfonylnitrene compound. It is to beunderstood that this compound can be substituted with R and R₁ asdefined above.

Nitrobenzene is chlorosulfonated at the meta position when contactedwith chlorosulfonic acid to form meta-nitrobenzenechlorosulfonic acid.The chlorosulfonic acid is then converted to the sulfonylazide byreacting with sodium azide in a solution of aqueous methanol. Thecompounds of FIG. 11 and the materialnitroaromatic sulfonyl nitreneintermediate compositions are prepared by irradiating the azide when incontact with a material susceptible to attack by a nitrene. If properadherence between the material and the aromatic sulfonylazide isdifficult to maintain prior to and during the course of irradiation, aswith polyethylene, for example, an inert carrier such as FluorolubeS-30, a perfluorinated mineral oil obtained from Hooker ChemicalCompany, can be employed in order to maintain the appropriate contact.

The types of radiation which can be employed to convert the azide to thenitrene include ultraviolet, ultrasound, and X-ray. Generally, anyradiation which promotes removal of free nitrogen from an azide can beemployed. The irradiation time should be of sufficient length to promotethe removal of nitrogen from the azide but of insufficient length tocause alteration of the material's desirable characteristics. Thisirradiation time may vary from an hour or more to a mere "flash"photolysis.

After formation of the aromatic sulfonylnitrene material composition,the nitro grouping on the aromatic nucleus is conveniently reduced to anamino group by a reduction involving metals, for example, stannouschloride in hydrochloric acid. Sodium thiosulfate in sodium hydroxide iseffective as well.

The amino grouping is then diazotized. Nitrous acid prepared in situ bythe reaction of sodium nitrite and hydrochloric acid is a suitablereagent for the conversion. Once the diazonium salt is formed, it can becoupled with a substance which possesses antithrombotic or anticlottingactivity such as aforementioned. Standard diazonium salt couplingreaction conditions can be employed. The thrombogenic or clottingactivity of the material is inhibited by the coating affixed to thematerial by way of the aromatic sulfonyl nitrene molecule.

If compounds of FIG. 111 are desired, a nitroaromatic is reduced to anamino by way of the methods previously stated. The amino group is thendiazotized with nitrous acid and the resulting diazonium salt coupledwith an antithrombotic or anticlotting substance as aforementioned. Thisproduct is then reacted in turn with chlorosulfonic acid and sodiumazide. This intermediate compound is then contacted with a materialwhich is susceptible to interaction with a sulfonyl nitrene and thenirradiated. The thrombogenic or clotting activity of the material isinhibited by the coating affixed to the material by way of the aromaticsulfonylnitrene.

The actual bonding of the aromatic sulfonyl nitrene to the material isdifficult to identify since conventional means of crystallization andanalysis are not applicable to the system of this invention. Because ofthe well-known ability of a nitrene to insert into a carbon-hydrogenbond, see D. Breslow in "Nitrenes", Lwowski, Ed. Interscience, N.Y.,1970., and the absence of any reactive groups in polymers such aspolyethylene and polypropylene, the most reasonable assignment for thereaction product is that of a sulfonamide.

The following examples are not intended to limit but only to illustratethe invention:

EXAMPLE 1

A solution of 0.6 gram each of m- and p-nitrobenzenesulfonylazide, thecombination of isomers selected so as to reduce crystallinity, and 1.2milliliters of Fluorolube S-30 oil in 12 milliliters of acetone isprepared. A fifteen cm² sheet of polyethylene, showing no absorption inthe ultraviolet down to 230 nm., is sprayed with one-half of thissolution. The sheet is covered with a Vycor plate, an ultraviolettransparent quartz glass, and irradiated for one hour under a lowpressure mercury lamp. The sheet is then rinsed with acetone, sprayedwith the remaining solution and irradiated with the low pressure mercurylamp for another hour.

The sheet is rinsed with acetone and stirred at room temperature with10.0 gram of stannous chloride in 100 milliliters concentrated HCl for 4hours. The sheet is then removed, rinsed with water, stirred for fifteenminutes with 1N sodium hydroxide and rinsed again with water.

The sheet is then exposed for two minutes to an ice cold solution ofnitrous acid prepared from fifty milliliters of 2.5N HCl and 5 grams ofsodium nitrite in twenty milliliters of water. Following a brief rinsewith ice water, the sheet is immersed for two minutes in a 10% aqueoussolution of the monopotassium salt of 8-amino-1-naphthol-5,7-disulfonicacid. This couples the dye to a diazonium salt. The sheet is then washedwith acetone and dried and subjected to ultraviolet analysis once more.This sample shows an ultraviolet absorption bond at 250 nm whichdemonstrates the attachment of a chromaphore..

EXAMPLE 2

A suspension of 3.8 grams of m-toluidine in four millilitersconcentrated HCl and 20 milliliters of water is cooled in anice-methanol bath. 2.3 grams of sodium nitrite in a minimum amount ofwater is added. After five minutes, this solution is added to 10 gramsof 8-amino-1-naphthol-5,7-disulfonic acid in fifty milliliters of water.The fine precipitate is collected on a filter and vacuum dried to give adark red powder. Infra red and ultraviolet analysis show this powder tobe the coupling product.

1.73 grams of the above prepared powder is added to ten milliliters ofchlorosulfonic acid. After 6 hours of vigorous stirring, the mixture ispoured into a small amount of ice water. The precipitatedchlorosulfonate, as identified by I. R., is collected on a filter andvacuum dried.

Two grams of sodium azide in ten milliliters of H₂ O is added to asolution of the sulfonyl chloride. Small aliquots are removed and workedup to monitor for appearance of sulfonyl azide. (IR_(max) 2250 cm⁻¹).When the reaction is complete the cosolvent is removed under vacuum andthe residue suspended in a small amount of ice water. The residualreddish solid is then collected on a filter.

The powder is coated on the polyethylene and irradiated in the samemanner as Example 1, the nitrene being formed and apparently insertinginto a carbon-hydrogen bond of the material.

EXAMPLE 3

The following procedures for testing the antithrombotic activity of acoated material is employed in later examples.

A Y-tube extracorporeal shunt is placed between the carotid artery andthe jugular vein of a rabbit. When a treated polymer, which can beshaped into a thin flexible sheet, is under study, a small piece of thetreated polymer is placed in one arm of the Y-tube. The other arm issimilarly lined with a portion of the non-treated polymer for use as acontrol. When a treated polymer which is relatively inflexible isstudied, a straight section of the treated polymer is connected directlyinto the carotid jugular by-pass. An untreated control section is placedin tandem.

The animal is given 100 μ/kg. of heparin and the shunt is allowed toflow for one hour. The Y-tube is disconnected, rinsed gently in Tyrodes'solution followed by distilled water and ethanol. The materials are thenstained with Luxol Blue, a dye, visually observed and photographed. Theplatelet deposits on the control and treated polymer are then compared.

EXAMPLE 4

The following procedure for testing the anticlotting activity of acoated material is employed.

A polyethylene or polypropylene test tube is coated with the desiredanticlotting substance using the methods employed in this disclosure.Whole blood is collected into these tubes and allowed to clot andassayed by the tilting tube method. Platelet involvement in thisreaction can be minimized by adding an inhibitor of platelet aggregationwhich does not interfere with blood coagulation. A control tube withouta coating and a control tube with a coating of the nitro or aminoaromatic sulfonyl nitrene are tested at the same time.

Differences in clotting time from the control tube and the treated tubeare measured.

EXAMPLE 5

Following the procedure of Example 1, polypropylene tubing was madenonthrombotic with 8-amino-1-naphthol-5,7-disulfonic acid. The treatedtube was then tested for nonthrombogenicity in accordance with themethod of Example 3. Virtually no deposit of platelets was observed onthe treated polypropylene but substantial deposits were on the control.

EXAMPLE 6

Following the procedure of Example 1, polyethylene, Dacron, and latexwere made nonthrombogenic with 8-amino-1-naphthol-5,7-disulfonic acid.The treated polyethylene surface was almost devoid of platelets. Thetreated latex showed substantially less deposit than the untreatedcontrol. Since the deposits on the treated tubing were primarily acrossthe lateral portion of the tubing, the deposits may be due to cracksproduced by the inversion of the tubing for treatment.

With regard to the Dacron employed, it also appears to have lessplatelet deposits upon it than upon the control. However, a significantportion of the platelet deposits on the treated Dacron may be due to theSilastic backing on the Dacron, since previous work has shown that thetreatment of Silastic with the coating material causes the Silastic todeteriorate, thereby causing greater platelet deposition.

EXAMPLE 7

In accordance with the method of Example 1, a polyethylene sheet iscontacted with the m-nitro aromatic sulfonyl azide and irradiated. Theresulting treated sheet is tested for nonthrombogenecity in accordancewith the method of Example 3. Upon inspection, substantially the samenumber of platelets are on both the treated and the control sheets.Consequently, a nonthrombogenic effect has not been imparted to thetreated polyethylene by the nitro aromatic sulfonyl nitrene.

In like manner, p-amino aromatic sulfonyl azide is contacted with apolyethylene sheet and irradiated. The resulting treated sheet is testedfor nonthrombogenecity as in Example 3. A nonthrombogenic effect is notimparted to the treated polyethylene by the amino aromatic sulfonylnitrene.

These results support the interpretation that it is the antithromboticsubstance coupled to the diazonium salt which inhibits the thromboticeffects of the polymeric material.

The small quantity of heparin administered to the subject rabbit doesnot appear to alter the results of the experiments. Heparin interfereswith fibrin formation and does not inhibit the deposition of platelets.Furthermore, since the same animal is used for evaluating both controland treated materials, any effect of heparin appears to be nullified.

We claim:
 1. A composition which comprises a sulfonyl aromatic diazoniumsalt chemically bonded at the sulfonyl grouping through anitrogen-containing bond to a material which is susceptible tointeraction with a nitrene which induces deposition of blood cells andfibrin when in contact with mammalian blood.
 2. A composition whichcomprises ##STR4## wherein X is nitro or amino, R and R₁ are the same ordifferent and are each selected from the group consisting of hydrogen,halogen, and alkyl of one to four carbon atoms, inclusive; and C is amaterial which is susceptible to interaction with an aromatic nitrenewhich induces deposition of blood cells and fibrin when in contact withmammalian blood.
 3. A composition in accordance with claim 2 wherein Xis nitro.
 4. A composition in accordance with claim 2 wherein X isamino.
 5. A composition which comprises a substituted aromatic compoundthat inhibits the thrombotic or clotting activity of a materialsusceptible to interaction with a sulfonylnitrene, said material beingone which induces deposition of blood cells and fibrin when in contactwith mammalian blood, said substituted aromatic compound is chemicallybonded to a phenyl, monosubstituted phenyl, or disubstituted phenyl,which is chemically bonded to a sulfonyl group through anitrogen-containing chemical bond to said material.
 6. A composition inaccordance with claim 5 wherein the material is a synthetic or naturalpolymer with a plurality of carbon-hydrogen bonds.
 7. A composition inaccordance with claim 6 wherein the nitrogen-containing bond to thematerial is a covalent bond between the nitrogen and a carbon atom ofthe material.
 8. A composition in accordance with claim 7 wherein thephenyl, monosubstituted phenyl or disubstituted phenyl is ##STR5##wherein R and R₁ are the same or different and are selected from thegroup consisting of hydrogen, halogen, and alkyl of one to four carbonatoms, inclusive.
 9. A composition in accordance with claim 6 whereinthe material is polyethylene or polypropylene.